Processing of intermediates in recombination and DNA repair: identification of a new endonuclease that specifically cleaves Holliday junctions.

The formation and subsequent resolution of Holliday junctions are critical stages in recombination. We describe a new Escherichia coli endonuclease that resolves Holliday intermediates by junction cleavage. The 14 kDa Rus protein binds DNA containing a synthetic four-way junction (X-DNA) and introduces symmetrical cuts in two strands to give nicked duplex products. Rus also processes Holliday intermediates made by RecA into products that are characteristic of junction resolution. The cleavage activity on X-DNA is remarkably similar to that of RuvC. Both proteins preferentially cut the same two strands at the same location. Increased expression of Rus suppresses the DNA repair and recombination defects of ruvA, ruvB and ruvC mutants. We conclude that all ruv strains are defective in junction cleavage, and discuss pathways for Holliday junction resolution by RuvAB, RuvC, RecG and Rus.     

Structural basis for isozyme-specific regulation of electron transfer in nitric-oxide synthase

Three nitric-oxide synthase (NOS) isozymes play crucial, but distinct, roles in neurotransmission, vascular homeostasis, and host defense, by catalyzing Ca(2+)/calmodulin-triggered NO synthesis. Here, we address current questions regarding NOS activity and regulation by combining mutagenesis and biochemistry with crystal structure determination of a fully assembled, electron-supplying, neuronal NOS reductase dimer. By integrating these results, we structurally elucidate the unique mechanisms for isozyme-specific regulation of electron transfer in NOS. Our discovery of the autoinhibitory helix, its placement between domains, and striking similarities with canonical calmodulin-binding motifs, support new mechanisms for NOS inhibition. NADPH, isozyme-specific residue Arg(1400), and the C-terminal tail synergistically repress NOS activity by locking the FMN binding domain in an electron-accepting position. Our analyses suggest that calmodulin binding or C-terminal tail phosphorylation frees a large scale swinging motion of the entire FMN domain to deliver electrons to the catalytic module in the holoenzyme.     

Neuropeptide delivery to synapses by long-range vesicle circulation and sporadic capture

Neurotransmission requires anterograde axonal transport of dense core vesicles (DCVs) containing neuropeptides and active zone components from the soma to nerve terminals. However, it is puzzling how one-way traffic could uniformly supply sequential release sites called en passant boutons. Here, Drosophila neuropeptide-containing DCVs are tracked in vivo for minutes with a new method called simultaneous photobleaching and imaging (SPAIM). Surprisingly, anterograde DCVs typically bypass proximal boutons to accumulate initially in the most distal bouton. Then, excess distal DCVs undergo dynactin-dependent retrograde transport back through proximal boutons into the axon. Just before re-entering the soma, DCVs again reverse for another round of anterograde axonal transport. While circulating over long distances, both anterograde and retrograde DCVs are captured sporadically in en passant boutons. Therefore, vesicle circulation, which includes long-range retrograde transport and inefficient bidirectional capture, overcomes the limitations of one-way anterograde transport to uniformly supply release sites with DCVs.     

Mouse divalent metal transporter 1 is a copper transporter in HEK293 cells

Divalent Metal Transporter 1 (DMT1) is an apical Fe transporter in the duodenum and is involved in endosomal Fe export. Four protein isoforms have been described for DMT1, two from mRNA with an iron responsive element (IRE) and two from mRNA without it. The sets of two begin in exon 1A or 2. We have characterized copper transport using mouse 2/?IRE DMT1 during regulated ectopic expression. HEK293 cells carrying a TetR:Hyg element were stably transfected with pDEST31 containing a 2/?IRE construct. ⁶⁴Cu¹⁺ incorporation in doxycycline treated cells exhibited 18.6 and 30.0-fold increases in Cu content, respectively when were exposed to 10 and 100 μM of extracellular Cu. Cu content was ~4-fold above that of parent cells or cells carrying just the vector. ⁶⁴Cu uptake in transfected cells pre-incubated with 5 μM of Cu-His revealed a Vmax and Km of 11.98 ± 0.52 pmol mg protein^?1 min^?1 and 2.03 ± 0.03 μM, respectively. Doxycycline-stimulated Cu uptake was linear with time. The rates of apical Cu uptake decreased and transepithelial transport increased when intracellular Cu increased. The optimal pH for Cu transport was 6.5; uptake of Cu was temperature dependent. Silver does not inhibit Cu uptake in cells carrying the vector. In conclusion, Cu uptake in HEK293 cells that over-expressed the 2/?IRE isoform of DMT1 transporter supports our earlier contention that DMT1 transports Cu as Cu¹⁺.     

Microgeographic socio-genetic structure of an African cooperative breeding passerine revealed: integrating behavioural and genetic data

Dispersal can be motivated by multiple factors including sociality. Dispersal behaviour affects population genetic structure that in turn reinforces social organization. We combined observational information with individual-based genetic data in the Karoo scrub-robin, a facultative cooperatively breeding bird, to understand how social bonds within familial groups affect mating patterns, cause sex asymmetry in dispersal behaviour and ultimately influence the evolution of dispersal. Our results revealed that males and females do not have symmetrical roles in structuring the population. Males are extremely philopatric and tend to delay dispersal until they gain a breeding position within a radius of two territories around the natal site. By contrast, females dispersed over larger distances, as soon as they reach independence. This resulted in male neighbourhoods characterized by high genetic relatedness. The long-distance dispersal strategy of females ensured that Karoo scrub-robins do not pair with relatives thereby compensating for male philopatry caused by cooperation. The observed female-biased strategy seems to be the most prominent mechanism to reduce the risk of inbreeding that characterizes social breeding system. This study demonstrates that tying together ecological data, such as breeding status, determining social relationships with genetic data, such as kinship, provides valuable insights into the proximate causes of dispersal, which are central to any evolutionary interpretation.     

Sampling efficacy for the red imported fire ant Solenopsis invicta (Hymenoptera: Formicidae)

Cost-effective detection of invasive ant colonies before establishment in new ranges is imperative for the protection of national borders and reducing their global impact. We examined the sampling efficiency of food-baits and pitfall traps (baited and nonbaited) in detecting isolated red imported fire ant (Solenopsis invicta Buren) nests in multiple environments in Gainesville, FL. Fire ants demonstrated a significantly higher preference for a mixed protein food type (hotdog or ground meat combined with sweet peanut butter) than for the sugar or water baits offered. Foraging distance success was a function of colony size, detection trap used, and surveillance duration. Colony gyne number did not influence detection success. Workers from small nests (0- to 15-cm mound diameter) traveled no >3 m to a food source, whereas large colonies (>30-cm mound diameter) traveled up to 17 m. Baited pitfall traps performed best at detecting incipient ant colonies followed by nonbaited pitfall traps then food baits, whereas food baits performed well when trying to detect large colonies. These results were used to create an interactive model in Microsoft Excel, whereby surveillance managers can alter trap type, density, and duration parameters to estimate the probability of detecting specified or unknown S. invicta colony sizes. This model will support decision makers who need to balance the sampling cost and risk of failure to detect fire ant colonies.     

Identifying conservation hotspots in non-breeding areas: a case study of Lake Sturgeon (<Emphasis Type="Italic">Acipenser fulvescens</Emphasis>) in the Great Lakes

Limited conservation resources necessitate the prioritization of management efforts and one of the mechanisms for prioritization is the identification of hotspots. For some species of conservation concern, hotspots have been delineated, often using individual abundance as the metric. However, areas that attract individuals from multiple breeding populations may be a higher priority for conservation. These areas may have more abundant resources or habitat quality, and multiple breeding populations simultaneously benefit from conservation actions in these areas. We identified non-spawning aggregations of Lake Sturgeon Acipenser fulvescens in lakes Huron and Erie of the Great Lakes. The spawning period in Lake Sturgeon only represents a brief portion of their life and conservation goals need to include non-spawning areas. Based on 12 microsatellite loci, we determined the population of origin of individuals in the aggregations and computed a modified population-level Simpson’s diversity index (pD). Using this criterion, two hotspots were identified. The North Channel of Lake Huron had the highest diversity of represented populations (pD = 3.14). The second identified hotspot was Saginaw Bay in Lake Huron (pD = 1.78). Both hotspots had evidence of long-distance movement into the area. Monitoring of aggregations should continue to assess temporal variability in hotspot delineation.     

Levels of extra‐pair paternity are associated with parental care in penduline tits (Remizidae)

In most passerine birds, individuals attempt to maximize their fitness by providing parental care while also mating outside their pair bond. A sex‐specific trade‐off between these two behaviours is predicted to occur, as the fitness benefits of extra‐pair mating differ between the sexes. We use nest observations and parentage analysis to reveal a negative association between male care and the incidence of extra‐pair paternity across three species of penduline tit (Remizidae). This provides evidence of a trade‐off between these two behaviours, possibly due to the devaluing of paternal care by extra‐pair offspring.